Bacterial H-NS contacts DNA at the same irregularly spaced sites in both bridged and hemi-sequestered linear filaments

Abstract

SUMMARYGene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD–DNA contacts in linear and bridged filaments are uncertain. To characterize the contacts that silence transcription by RNA polymerase, we combined ·OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ~10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching in which linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contact sites.HighlightsTethered-nuclease mapping (TEN-map) of H-NS DNA-binding domains detects DNA contactsBridged and linear H-NS filaments use the same DNA contact sitesH-NS–DNA contacts are unevenly spaced with ~10 bp average separationAT-steps, minor groove width, and electrostatic potential best predict contact sites